Aging alters the cell cycle control and mitogenic signaling responses of human hematopoietic stem cells.

Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor-stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.

A topological view of human CD34+ cell state trajectories from integrated single-cell output and proteomic data.

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

Discovery of 2'-hydroxychalcones as autophagy inducer in A549 lung cancer cells.

A series of 2'-hydroxychalcone derivatives was synthesized and the effects of all the compounds on growth of A549 lung cancer cell were investigated. The results showed that all compounds had inhibitory effects on the growth of A549 lung cancer cells and compound possessed the highest growth inhibitory effect and induced autophagy of A549 lung cancer cells.

A highly sensitive fluorescent probe based on simple pyrazoline for Zn2+ in living neuron cells.

We develop a pyrazoline-based fluorescent sensor for biological Zn(2+) detection. The sensor shows good binding selectivity for Zn(2+) over competing metal with 40-fold fluorescence enhancement in response to Zn(2+). The new probe is cell-permeable and can be used to detect intracellular zinc ions in living neuron cells.

Sensitive and effective imaging of carbon monoxide in living systems with a near-infrared fluorescent probe.

CO, a gas molecule that is harmful to living organisms, has a high affinity with hemoglobin, which will cause severe hypoxia. However, in recent years, researchers have discovered that endogenous CO, similar to NO, is one of the messenger molecules, which has a certain regulatory effect in many physiological and pathological processes in the respiratory system, cardiovascular system, and nervous system. Therefore, it is urgent to explore an effective method to monitor the role of CO under physiological and pathological conditions. Herein, we designed and synthesized a near-infrared small-molecule fluorescent probe for the detection of CO in living cells. In this design, a two-site BODIPY dye was introduced as the fluorophore, and the allyl chloroformate part as the CO reactive group. The probe displays excellent sensitivity, selectivity, and a good linear relationship to CO. Furthermore, it shows good biocompatibility and low cytotoxicity. This probe has been successfully applied to the detection of CO in a variety of cells. The developed fluorescent probe can serve as a potential molecular imaging tool for in vivo imaging and detection of CO.

Development of an ultrafast fluorescent probe for specific recognition of hypochlorous acid and its application in live cells.

Hypochlorous acid (HOCl), a highly potent oxidant of reactive oxygen species, plays critical roles in many physiological and pathological processes. In this work, a novel coumarin-based fluorescent probe, Cou-HOCl, was prepared for the detection of HOCl. The probe exhibited good selectivity over other analytes, excellent sensitivity with a detection limit of 16 nM, and fast response within 5 s. And further study demonstrated that the probe could be used not only to image exogenous HOCl in various cells, but also to determine the fluctuating levels of HOCl in macrophage cells during inflammation.

Single-cell analysis identifies a CD33+ subset of human cord blood cells with high regenerative potential.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

A new turn-on fluorescence probe for Zn(2+) in aqueous solution and imaging application in living cells.

We designed and synthesized a new pyrazoline-based turn-on fluorescence probe for Zn(2+) by the reaction of chalcone and thiosemicarbazide. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy. The probe (L) exhibits high selectivity and sensitivity for detecting Zn(2+) in buffered EtOH/HEPES solution (EtOH/HEPES=1/1, pH 7.2) with 80-fold fluorescence enhancement, which is superior to previous reports. Job's plot analysis revealed 1:1 stoichiometry between probe L and Zn(2+) ions. The association constant estimated by the Benesi-Hildebrand method and the detection limit were 3.92×10(3)M(-1) and 5.2×10(-7)M, respectively. A proposed binding mode was confirmed by (1)H NMR titration experiments and density functional theory (DFT) calculations. The probe is cell-permeable and stable at the physiological pH range in biological systems. Because of its fast response to Zn(2+), the probe can monitor Zn(2+) in living cells. Moreover, the selective binding of L and Zn(2+) was reversible with the addition of EDTA in buffered EtOH/HEPES solution and Zn(2+) could be imaged in SH-SY5Y neuron cells.